We propose to use mutant enzymes for gene therapy of human cancer, both to ablate tumors and to protect bone marrow during chemotherapy. We have established techniques for creating new mutant enzymes by coupling random nucleotide mutagenesis with genetic selection for enzymes with altered substrate specificities. We will use this technology to create mutant enzymes with desired properties for cancer gene therapy, and will test their efficacity in a variety of experimental systems. We will pursue four strategies that target different steps in DNA metabolism. We have established a selection system to identify mutant thymidylate synthases that are resistant to 5-fluorouracil, to be used for protection of bone marrow during therapy with this commonly employed agent. We have identified mutant DNA methyltransferases that are more active than the wild type, and are resistant to the inhibitor benzylguanine, to be used to increase the resistance of bone marrow precursor cells to alkylating agents. We will evaluate whether mutant DNA polymerase beta's can serve to enhance replication bypass and thereby increase the tolerance of bone marrow cells for unrepaired lesions that block DNA synthesis. We have established a large library of mutant herpes thymidine kinases that preferentially phosphorylate nucleoside analogs. These mutant thymidine kinases sensitize mammalian cells to the lethal effects of gancyclovir and acyclovir to a greater extent than the wild type enzyme, and will be tested for efficacity in tumor ablation.